Structural Transitions of the Conserved and Metastable Hantaviral Glycoprotein Envelope

Abstract

Hantaviruses are zoonotic pathogens that cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in northeastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.IMPORTANCE Although outbreaks of Korean hemorrhagic fever were first recognized during the Korean War (1950 to 1953), it was not until 1978 that they were found to be caused by Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Here, we describe the crystal structure of HTNV envelope glycoprotein Gn, an integral component of the Gn-Gc glycoprotein spike complex responsible for host cell entry. HTNV Gn is structurally conserved with the Gn of a genetically and geographically distal hantavirus, Puumala virus, indicating that the observed α/β fold is well preserved across the Hantaviridae family. The combination of our crystal structure with solution state analysis of recombinant protein and electron cryo-microscopy of acidified hantavirus allows us to propose a model for endosome-induced reorganization of the hantaviral glycoprotein lattice. This provides a molecular-level rationale for the exposure of the hydrophobic fusion loops on the Gc, a process required for fusion of viral and cellular membranes.

Keywords: X-ray crystallography; bunyavirus; cryo-EM; hantavirus; host cell infection; structural biology; viral glycoprotein; virus structure.

FIG 1

Phylogeny and crystal structure of HTNV Gn ectodomain. (A) A maximum-likelihood phylogeny of 42 hantaviral Gn glycoprotein sequences separates hantavirus species according to host reservoir. The three clades of hantaviruses borne by rodents are annotated in yellow, green, and blue. Hantaviruses carried by shrews and moles are annotated in purple, and Longquan virus, isolated from bats, is annotated in white. The scale bar indicates amino acid substitutions per site. (B) The organization of the HTNV glycoprotein precursor (above) and crystal structure of the HTNV Gn ectodomain to 2.15-Å resolution (below). The schematic was produced with DOG (60) with the crystallized region of the Gn indicated by a bar colored as a rainbow. The signal peptide (SP), transmembrane domains (TM), the hydrophobic region preceding the WAASA cleavage site (TM′), intraviral domains (IV), and WAASA signal peptidase cleavage site are annotated. Putative N-linked glycosylation sequons are labeled above the schematic (pink pins). The structure is presented as a cartoon and colored as a rainbow ramped from blue (N terminus) to red (C terminus). Disordered regions comprising residues 190 to 197 and residues 281 to 289 are highlighted (dotted lines, green and orange, respectively). The crystallographically observed glycan at Asn134 is shown as pink sticks. (C) Structural comparison of HTNV Gn and PUUV Gn. Overlay of HTNV Gn and PUUV Gn, colored as a rainbow and in gray, respectively (ribbon representation), is shown on the left. At right is the HTNV Gn with root mean square (RMS) deviation of equivalent residues between PUUV Gn mapped onto the Cα trace. The tube radius and color represent the RMS deviation (ramped from blue to red). Regions with high deviations between PUUV Gn and HTNV Gn structures are thick and red. Regions with low deviations are thin and blue.

FIG 2

Fitting of HTNV Gn into the TULV glycoprotein spike localizes the Gn to tetrameric membrane distal lobes. (A) The TULV glycoprotein spike (Electron Microscopy Data Bank [EMDB] accession no. EMD-3364) was partitioned into five unique segments, as previously described (12): two globular membrane-distal volumes (segment A and A′, light and dark blue, respectively), two elongated volumes (segment B and B′, pink and salmon, respectively), and a central stalk (segment C, gray), using Segger (58). Fitting analysis, using the fit-to-segments function of Segger, reveals that HTNV Gn most likely localizes to the membrane-distal volumes, as shown in the plot (right) that illustrates the goodness of fit (density occupancy score) of HTNV Gn into the density segments. The two highest-scoring fitting outcomes for Gn are shown within the globular membrane-distal volumes of TULV (12) (B) and HTNV (10) (C) cryo-EM reconstructions.

FIG 3

The acidic tetramer of HTNV Gn presents an oligomeric conformation distinct from that predicted at neutral pH. (A) Structure of the crystallographically observed HTNV Gn tetramer. Three protomers are colored in shades of gray in a surface representation. The fourth Gn protomer is shown as a cartoon and colored as described in the legend to Fig. 1B. Insets show that the interaction interface is facilitated by the Tyr82-Phe96 β-hairpin loop, which interlocks into a pocket formed in the adjacent protomer. (B) Comparison of the tetramers formed from the two highest scoring fits, presented in Fig. 2, with that of the crystallographically observed acidic tetramer. On the left-hand side, the two highest scoring fits are shown (cartoon representation) with the membrane-distal volumes of the TULV spike reconstruction (EMDB accession no. EMD-3364) shown as a gray background. The right-hand side shows the acidic tetramer placed in the same position, revealing that that the span of the assembly is approximately 50 Å less than the width of the tetramer observed at physiological pH. (C) Analytical ultracentrifugation analysis reveals that HTNV Gn forms higher-order oligomeric states in solution upon acidification, including putative dimers, trimers, and tetramers. A sedimentation coefficient distribution plot shows populations of HTNV Gn at pH 7.0 (in gray) and 4.5 (in black). Experimentally derived sedimentation coefficient values (Svedberg units [S]) are shown above each peak. For comparison, theoretical S values, which were calculated using the crystallographically observed acidic tetramer and derivative monomer, dimer, and trimer subcomponents, are shown with equivalent subunit representations to the right. The range of values represents variable possible glycosylation states of the Gn protein: the first value is calculated without glycans modeled, and the second value is calculated with high-mannose-type glycans (61) modeled at N-linked glycosylation sites as rigid side chains. This analysis revealed that the experimental peaks at 3.5 to 3.9S, 5.2S, 7.2S, and 8.2S closely approximate the expected migration of a monomer, dimer, trimer, and tetramer, respectively. In addition, minor populations of larger HTNV Gn oligomers were detected. Theoretical S values were calculated using SoMo (62–64) with the optimal overlap bead model approach and hydrodynamic parameter determination with ZENO (62).

FIG 4

Cryo-electron microscopy analysis reveals the metastability of the hantavirus envelope. (A and B) Representative cryo-electron micrographs of TULV after incubation at pH 7 and pH 5, respectively. Scale bar, 100 nm. (C and D) Close-ups of the areas indicated in panels A and B with the regular lattice of Gn-Gc spikes indicated with arrows in panel C. No regular lattice is visible in the virions incubated at pH 5. (E and F) Representative 2D class averages of the virion surface at pH 7 and pH 5. Density has been inverted so that white corresponds to the virion. (G) An averaged density profile of the images in panels E and F indicating the average distribution of density perpendicular to the membrane at pH 7 and pH 5. Densities corresponding to the lower membrane leaflet (LL), the upper membrane leaflet (UL), and Gn-Gc spikes are indicated. a.u., arbitrary units.

FIG 5

A model of pH-dependent conformational changes to the Gn and Gc glycoproteins during endocytosis, prior to fusion. At left is a side-view schematic of the mature hantavirus envelope, based upon a previously reported reconstruction of the TULV glycoprotein spike (EMDB accession no. EMD-3364). The globular Gn glycoprotein (blue), Gc glycoprotein (salmon), C terminus of the Gn-glycoprotein (gray), and putative location of the Gc fusion loops (FL; buried in the Gn-Gc interface), are indicated. Disassembly of the Gn-Gc spike complex (right panel) and the formation of the acidic Gn tetramer could create the space necessary for trimerization of the Gc glycoprotein with the fusion loops facing outwards to enable contact with target cellular membranes. The Gc trimer shown in the schematic is based on the HTNV Gc crystal structure (PDB accession number 5LJZ) (14), with domain III rotated 90° to model an extended class II fusion protein trimer that has been suggested to exist at low pH, prior to membrane fusion (35, 39). The width of the putative Gn tetramers formed under both pH-neutral and acidic conditions is annotated.